Cancer is a disease in which normal body cells are changed, becoming able to multiply without regard to normal cellular restraints and to invade and colonize areas of the body normally occupied by other cells. See B. Alberts et al., Molecular Biology of the Cell 1255-1294 (3d ed. 1994). According to the American Cancer Society, one-half of all American men and one-third of all American women will at some point in their lives develop cancer.
Due to the ability of cancer cells to spread and rapidly proliferate, it is difficult to treat cancer patients by attempting to selectively kill cancerous cells. Some have compared the difficulty of this task to the difficulty of completely ridding a garden of weeds. As with weeds, if only a few cancer cells are left untouched by treatment, they may again spread throughout the body, causing a recurrence of the disease. See id. at 1267. Current treatments for cancer include surgery and therapies using chemicals and radiation. The effectiveness of these treatments is often limited, however, since cancer cells that have spread from the original tumor site may be missed by surgery and radiation, and since chemical treatments which kill or disable cancer cells are often capable of causing similar damage to normal cells. See id.
Hope for better treatments for cancer focuses on obtaining a better understanding of carcinogenesis—the series of events which transforms a normal cell into a cancer cell. It is hoped that such an understanding will help researchers and physicians direct treatments solely toward cancer cells or their precursors, thus preventing or treating cancer and avoiding damage to healthy body tissues. As more data is gathered on the transformation of a normal cell into a cancer cell, it is apparent that a number of genes and proteins can play a role in carcinogenisis. Additionally, there are many proteins whose activity has not been fully determined and that may play a role in carcinogenesis.
Diacylglycerol kinase (DGK) belong to one family of enzymes that are not completely understood. DGKs catalyze the phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA). Sakane, F. & Kanoh, Int J Biochem Cell Biol 29, 1139-1143 (1997); Topham, M. K. & Prescott, S. M. J Biol Chem 274, 11447-50 (1999). Both the substrate (DAG) and the product (PA) of the DGK reaction are key factors in intracellular signaling. For example, DAG activates protein kinase Cs (PKC) and some guanine nucleotide exchange factors such as RasGRP. Nishizuka, Y. Science233, 305-312 (1986).; Ebinu, J. O. et al. Science 280, 1082 (1998). The consumption of DAG by DGKs is thought to attenuate these actions, so DGKs are thought to terminate the activity of PKCs and other DAG-activated proteins. Conversely, by generating phosphatidic acid, DGKs may initiate a variety of cellular events. For example, PA has been reported to modulate a typical PKC isoforms, Ras-GAP, phosphatidylinositol (PI) 5-kinases (Moritz et al., 1992) and other signaling proteins, and PA is mitogen for a variety of cells. Exton, J. H. Physiological Reviews 77, 303 (1997).
It is unclear which functions attributable to DAG and PA reflect the actions of DGK, since phospholipase D (PLD) also releases PA, and DAG is also produced by PA phosphatase. Sakane, F. & Kanoh, Int J Biochem Cell Biol 29, 1139-1143 (1997); Topham, M. K. & Prescott, S. M. J Biol Chem 274, 11447-50 (1999). Exton, J. H. Physiological Reviews 77, 303 (1997). It is likely, however, that signaling lipids derived from each pathway—the PLC/DGK or PLD/PA phosphatase—have distinct functions by virtue of the parent lipids for each reaction. Hodgkin, M. N. et al. Trends Biochem Sci 23, 200-(1998). For example, the predominant substrate of PLD is phosphatidylcholine, which is composed primarily of saturated fatty acids, so the reaction product, phosphatidic acid, is also composed mostly of saturated fatty acids. Alternatively, DGKs are thought to phosphorylate DAG generated by phosphatidylinositol—specific phospholipase Cs. Since phosphatidylinositols are enriched in unsaturated fatty acids, DAG derived from this reaction is predominantly unsaturated, so the PA generated by DGK activity is composed mostly of unsaturated fatty acids. Id. And, there is evidence that DAG and PA, depending on their lipid composition, can differentially activate protein targets. For example, unsaturated DAG is a more potent activator of protein kinase Cs than is saturated DAG, while saturated PA species induce MAPK activation to a greater extent that unsaturated PAs. Thus, DGKs and PLDs likely influence distinct signaling events.
While most attention on PA signaling has been focused on the PLD reaction, PA generated by DGKs likely has signaling functions as well. Flores and colleagues identified a potential role for DGK-generated PA in T lymphocyte proliferation. Flores, I., et al. J Biol Chem 271, 10334-10340 (1996). They noted that when T lymphocytes were treated with IL-2, a growth signal, DKGα translocated to the perinuclear space. Using DGK inhibitors, they presented evidence that the PA produced by this isozyme was necessary for progression to S phase of the cell cycle, suggesting that PA generated by DKGα in this context had a signaling role. Additionally, Cutrupi et al demonstrated that active DKGα was required for hepatocyte growth factor induced migration of endothelial cells. Cutrupi, S. et al. EMBO J. 19, 4614-4622 (2000). Their data suggested that generation of PA by DKGα was necessary for the migration, but they could not identity the protein target of the phosphatidic acid.
As mentioned above, there are many proteins whose activity can be influenced by PA, so DGKs could regulate a variety of cellular events that are dependent on PA. Diacylglycerol kinases can also influence proteins regulated by DAG. In this case, however, DGKs are likely inhibitory because they terminate DAG signaling. Indeed, DGKζ, and not other DGK isotypes, inhibited the activity of RasGRP, a Ras guanyl nucleotide exchange factor (GEF) whose activity requires DAG. Topham, M. K. & Prescott, S. M. J. Cell Biol. 152 (2001). And, Nurrish et al presented evidence that a Caenorhabditis elegans DGK negatively regulated synaptic transmission by metabolizing DAG that would otherwise activate Unc-13, a protein activated by DAG that participates in neurotransmitter secretion. Nurrish, S., et al. Neuron 24, 231-242 (1999). Thus, by virtue of their enzymatic activity, DGKs can influence signaling events mediated by both DAG and PA. As these lipids can affect many protein targets, diacylglycerol kinases occupy an interesting biologic niche.
The DGK family is large and diverse. As with other enzymes in signaling pathways, such as PKC and PI-specific phospholipase C, mammalian DGKs are a family whose isozymes differ in their structures, patterns of tissue expression and catalytic properties. Sakane, F. & Kanoh, Int J Biochem Cell Biol 29, 1139-1143 (1997); Topham, M. Y & Prescott, S. M. J Biol Chem 274, 11447-50 (1999). Nine mammalian DGK isoforms have been identified. All of them contain a catalytic domain that is necessary for kinase activity. The DGK catalytic domains likely function similarly to the C3 regions of PKCs by presenting ATP as the phosphate donor. In addition to these domains, all DGKs have at least two cysteine-rich regions homologous to the C1A and C1B motifs of PKCs. DGKθ has three. These domains in DGKs are thought to present diacylglycerol for phosphorylation, but this has not been conclusively demonstrated.
In addition to these motifs, most DGKs have other structural domains that likely perform regulatory roles and are used to group the DGKs into the five subfamilies. Type I DGKs have calcium-binding EF hand motifs, making these isoforms calcium responsive. Diacylglycerol kinases having PH domains at their amino termini are grouped as type II. No specific function has been identified for these domains, but the PH domain of DGKδ can bind phosphatidylinositols (PIs). DGKδ also has at its C-terminus a sterile alpha motif (SAM). Its function is unclear, but SAM domains can be sites of protein—protein interactions. DGKε is a type III enzyme, and although it does not have any identifiable regulatory domains, it strongly prefers an arachidonoyl group at the sn-2 position making it the only DGK that has specificity toward acyl chains of DAG. This preference suggests that DGKε may be a component of the PI cycle that accounts for the enrichment of PI species with arachidonate. Type IV DGKs have a region homologous to the phosphorylation site domain of the MARCKS protein, and at their C-termini, four ankyrin repeats. Finally, DGKθ is a type V enzyme with three cysteine-rich domains and a PH domain. Their structural diversity and distinct expression patterns suggest that each isoform may perform a different function.
While many DGK isoforms have been identified in mammals, one or only a few DGK isoforms have been identified in organisms such as Caenorhabditis elegans, Drosophila melanogaster, and Arabidopsis thaliana. Topham, M. K. & Prescott, S. M. J Biol Chem 274, 11447-50 (1999). Moreover, at present, no DGK gene has been identified in yeast. The number and distribution of mammalian DGKs suggest that they have roles in processes specific to higher vertebrates, such as development, advanced neural functions, immune surveillance, and tumorigenesis. To date, however, there have been few studies of the specific functions of individual DGK isoforms.
Understanding the DGKs occupy crucial positions in the regulation of cellular signaling agents, it would thus be an improvement in the art to characterize the function of a DGK isoform. Specifically it would be an improvement in the art to characterize the function of a mammalian DGKδ enzyme. It would be an additional advancement to provide methods of screening for agents that inhibit the activity of the DGKδ enzyme. It would be a further advancement if such agents could be used to inhibit cell growth or inflamation.